microorganisms

Review
Purine Acquisition and Synthesis by Human
Fungal Pathogens
Jessica L. Chitty 1,2 and James A. Fraser 1, *
1 Australian Infectious Diseases Research Centre, School of Chemistry & Molecular Biosciences,
the University of Queensland, St Lucia, Queensland 4072, Australia; j.chitty@uq.edu.au
2 Institute for Molecular Bioscience, the University of Queensland, St Lucia, Queensland 4072, Australia
* Correspondence: j.fraser1@uq.edu.au; Tel.: +61-7-3365-4868

Academic Editor: Julianne Djordjevic

Received: 31 May 2017; Accepted: 6 June 2017; Published: 8 June 2017

Abstract: While members of the Kingdom Fungi are found across many of the world’s most hostile
environments, only a limited number of species can thrive within the human host. The causative
agents of the most common invasive fungal infections are Candida albicans, Aspergillus fumigatus,
and Cryptococcus neoformans. During the infection process, these fungi must not only combat the
host immune system while adapting to dramatic changes in temperature and pH, but also acquire
sufficient nutrients to enable growth and dissemination in the host. One class of nutrients required by
fungi, which is found in varying concentrations in their environmental niches and the human host, is
the purines. These nitrogen-containing heterocycles are one of the most abundant organic molecules
in nature and are required for roles as diverse as signal transduction, energy metabolism and DNA
synthesis. The most common life-threatening fungal pathogens can degrade, salvage and synthesize
de novo purines through a number of enzymatic steps that are conserved. While these enable them
to adapt to the changing purine availability in the environment, only de novo purine biosynthesis is
essential during infection and therefore an attractive antimycotic target.

Keywords: fungal pathogens; purines; nitrogen; degradation; salvage; synthesis

1. The Diversity of Fungi and the Environments They Inhabit

Federico Cesi, founder of the Accademia dei Lincei and a keen observer of his local environment, first
attempted the scientific classification of organisms. “Imperfect” plants, particularly fungi, fascinated
Cesi; his colleague Galileo Galilei constructed a microscope to help him observe these organisms in
great detail, and Cesi commissioned hundreds of drawings of mushroom species collected from Rome
and southern Umbria until his death in 1630 [1]. The Italian priest and botanist Pier Antonio Micheli
later continued Cesi’s classification. His most notable work Nova Plantarum Genera documented
1400 new “plant” species collected from around Europe, of which 900 were fungi or lichens and
included the first documented human fungal pathogen [2]. A few decades later, Swedish botanist and
zoologist Carl Linné made a significant contribution to modern taxonomy by classifying organisms
from around the globe in his seminal work Systema Naturae, although fungal species were poorly
addressed in this publication [3]. Almost 100 years later, mycologists Christian Hendrik Persoon
and Elias Magnus Fries addressed these shortcomings by classifying fungi sent by leading scientists
from around the world [4–6]. Combined, these extensive works that took place over four centuries
identified thousands of fungi that expanded well beyond Cesi’s Italian mushrooms to include more
diverse species inhabiting a wide range of environments. However, it was not until 1969 that the
Fungi were classified as their own kingdom and not a subset of plants [7,8]. With the aid of genomic
sequencing, the number of species identified now numbers over one million and these are believed to

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Microorganisms 2017, 5, 33 2 of 16

be just the tip of the iceberg, accounting for an estimated 5–7% of species with many environments
largely unsampled [9,10].
Species from the kingdom Fungi differ greatly in habitat, morphology and nutrient requirements.
As heterotrophs, these organisms digest organic molecules such as proteins, polysaccharides and
nucleotides, and are often found in nutrient rich environments. In contrast, many fungi can survive in
extreme conditions. Aspergillus sydowii inhabits deep-sea hydrothermal vents more than 700 meters
below sea level where temperatures reach almost boiling point [11]. Penicillium chrysogenum can
be found in the Atacama Desert, where it has been hyper arid for at least three million years [12].
Nadsoniella nigra var. hesuelica survives periodic freezing and thawing in Antarctica [13]. Other species
survive in a very different extreme environment—the human host —where fungal pathogens must be
heat tolerant, resilient to immune defenses and scavenge nutrients that can be difficult to acquire [14].
Of the fungal pathogens affecting humans, three pose the most consistent major threat worldwide:
Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans, causing aspergillosis, candidiasis
and cryptococcosis, respectively. Even with the best available antifungal treatment in developed
countries, low efficacy, toxicity and resistance is a major contributor to the high mortality associated
with these invasive fungal infections [15,16]. Each preferentially infects specific sites in patients, from
the lung in pulmonary aspergillosis to the brain in cryptococcal meningoencephalitis and the blood
stream in systemic candidiasis, yet all employ similar mechanisms to acquire sufficient nutrients to
survive and establish an infection [17–19].
Unlike C. neoformans and A. fumigatus, which are often found in soil, guano and decaying
matter [20–23], C. albicans is a commensal species, commonly found in the gastrointestinal tract
and on mucocutaneous surfaces [24,25]. While these environments are vastly different, one important
class of biological compounds consistently present are the purines.
To expel excess nitrogen, bird excreta contains high concentrations of the insoluble hetrocyclic
compound uric acid, a strategy that reduces water loss compared to the mammalian excretion of
nitrogen as its soluble derivative urea [26]. Fresh plant matter such as cauliflower has a purine
content of approximately 0.4 mg/g [27]; when living organisms, such as plants, decompose, purines
become enriched in soil and so are available to fungi such as C. neoformans and A. fumigatus [28].
Furthermore, the different tissues of the live human host also vary significantly in the context of purines.
The concentrations of purines in the gastrointestinal tract inhabited by C. albicans are dependent on
the host’s diet, whereas the cerebral spinal fluid to which C. neoformans disseminates is a particularly
purine-poor environment [29–31].

2. Purines and Their Role in the Cell

Purine, a term coined by Emil Fisher in 1884 after he synthesized a novel compound from “pure
urine”, is a molecule composed of one six and one five-membered nitrogen-containing ring fused
together. The addition of at least one phosphate to this molecule makes it a nucleotide, a biochemically
important component of the cell [32]. These molecules are essential to DNA and RNA biosynthesis,
energy metabolism and signal transduction, and are the most widely occurring nitrogen-containing
heterocycle in nature [33]. Purines are also predicted to have been amongst the first organic compounds
synthesized by abiotic chemistry on the early earth; adenine, the nucleobase of adenosine triphosphate
(ATP), is proposed to have been formed during the prebiotic era by the condensation of five hydrogen
cyanide molecules [34,35].
Microorganisms 2017, 5, 33 3 of 16
Microorganisms 2017, 5, 33 3 of 16

Figure 1. Blue represents enzymes found in C. albicans, A. fumigatus, and C. neoformans. Pink
Figure 1. Blue represents enzymes found in C. albicans, A. fumigatus, and C. neoformans. Pink
represents an enzyme found in both C. albicans and A. fumigatus. Green represents an enzyme found
represents an enzyme found in both C. albicans and A. fumigatus. Green represents an enzyme found in
in A. fumigatus only. Abbreviated enzyme names: PRP (Phosphoribosylpyrophosphate)
A. fumigatus only. Abbreviated enzyme names: PRP (Phosphoribosylpyrophosphate) amidotransferase,
amidotransferase, GAR (glycinamide ribotide) synthetase, GAR (phosphoribosyl-glycinamide)
GAR (glycinamide ribotide) synthetase, GAR (phosphoribosyl-glycinamide) transformylase, FGAM
transformylase, FGAM (formylglycinamidine-ribonucleotide) synthetase, AIR aminoimidazole
(formylglycinamidine-ribonucleotide)
ribotide) synthetase, AIR synthetase, AIR aminoimidazolecarboxylase,
(Phosphoribosylaminoimidazole) ribotide) synthetase,
SAICARAIR
(Phosphoribosylaminoimidazole) carboxylase, SAICAR (N-succinyl-5-aminoimidazole-4-carboxamide
(N-succinyl-5-aminoimidazole-4-carboxamide ribotide) synthetase, ADS (adenylosuccinate) lyase,
ribotide)
AICARsynthetase, ADS (adenylosuccinate) ribonucleotide)
(5-aminoimidazole-4-carboxamide lyase, AICAR transformylase,
(5-aminoimidazole-4-carboxamide
IMP (inosine
ribonucleotide) transformylase,
monophosphate) IMPADS
cyclohydrolase, (inosine monophosphate)
(adenylosuccinate) cyclohydrolase,
synthetase, ADSmonophosphate)
IMP (inosine (adenylosuccinate)
synthetase, IMP (inosine
dehydrogenase, monophosphate)
GMP (guanine dehydrogenase,
monophosphate) synthase. GMP (guanine monophosphate) synthase.
Microorganisms 2017, 5, 33 4 of 16

3. Purines as a Nitrogen Source

Defects in the degradation of purines for the excretion of excess nitrogen were first implicated
to play a role in disease in 1848 by Alfred Garrod. However, the biochemical process in a microbe of
degrading purines to acquire nitrogen required for growth was not studied until 1853 when Friedrich
Wohler investigated this process in an unidentified yeast species. Nitrogen is a major component
of a number of molecules, including amino acids, pyrimidines, and purines, and is essential for life.
Fungi are known for their ability to use a wide range of nitrogen sources via a range of catabolic
enzymes [36,37].
The process of utilizing purines as a nitrogen source proceeds via the degradation of xanthine
to uric acid by xanthine oxidase. Uric acid can then be sequentially degraded by a further five
enzymatic activities to produce ammonia (Figure 1). Depending on the fungal species, the entry point
for this degradation pathway varies. For example, during its evolution into a facultative anaerobe
Saccharomyces cerevisiae lost oxygen-dependent urate oxidase, but it can still use allantoin or allantoate
as a nitrogen source. In contrast, the fungal pathogen C. neoformans can utilize uric acid as a sole
nitrogen source [38–40].
The importance of maintaining a fully functional degradation pathway is not true for all
fungi; some plant colonizing species of fungi such as Piriformospora indica lack this ability [41].
The Pneumocystis pneumonia-causing fungus Pneumocystis jirovecii also lacks the catabolic enzymes
required for the degradation of purines, although this is the only human fungal pathogen known to do
so [42].

4. Salvaging Purines
As well as breaking down the purines obtained from the environment to serve as a nitrogen
source, fungi also scavenge these essential nutrients for metabolic processes. Small molecules such
as nucleotides are detected by plasma membrane-localized sensors to be transported across the
plasma membrane to be used in nucleotide biosynthesis [43]. A number of proteins have been
identified in fungi that transport purines. Three distinct nucleobase-specific transporter classes exist:
nucleobase-ascorbate transporter (NAT) families 1 and 2, the nucleobase cation symporter family 1
(NCS1) and the AzgA-like family. These are all secondary active transporters as they catalyze the
transport of two chemical species, a purine and a proton, in the same direction [44], Once scavenged
nucleotides are transported into the cell via these dedicated transporters, they are available for
incorporation into the salvage pathway. The principal enzymes responsible for the interconversion
of purines are hypoxanthine xanthine guanine phosphoribosyltransferase (HXGPT) and adenine
phosphoribosyltransferase, enzymes that transfer a 5-phosphoribosyl group to a purine to create the
corresponding nucleotide.

5. Synthesizing Purines
Fungi may not always be able to salvage sufficient purines from the environment; in many cases
nucleotides must be synthesized de novo from precursor molecules. Acquiring purines from the
environment is energetically favorable compared to de novo biosynthesis, which requires 14 enzymatic
activities and a number of cofactors for the magnesium-dependent generation of either ATP or
guanosine triphosphate (GTP) from phosphoribosyl pyrophosphate. Inosine monophosphate (IMP)
biosynthesis requires the acquisition of ammonia from two molecules of L-glutamine, ligation of
L -aspartate, and hydrolysis of four ATP molecules, with two 10-formyl-THF formyl donors. For the
synthesis of ATP from IMP (Figure 2), an additional L-aspartate molecule is ligated, and one GTP and
two ATP molecules are hydrolyzed. The synthesis of GTP from IMP (Figure 2) requires an additional
molecule of one L-glutamine, hydrolysis of three ATPs and the hydride transfer from one nicotinamide
adenine dinucleotide (NAD+ ) molecule. In total, in order for de novo synthesis to occur, 10 molecules
of ATP are hydrolyzed per molecule of AMP synthesized, and 11 for GMP [45,46]. It is estimated that
 Microorganisms 2017, 5, 33 5 of 16

these purines is the most energy efficient strategy, having an intact de novo purine synthesis pathway
is highly advantageous to fungi that inhabit environments with varying concentrations of purines
[46].
Microorganisms 2017, 5, 33 5 of 16
Some species of fungi have lost the ability to synthesize purines and rely solely on the salvage
of nucleotides from their environment. The Microsporidia have lost a number of enzymatic activities
required
10 for the de are
7 ATP molecules novoused
synthesis of purines,
per second per S.meaning
cerevisiaethe complete
cell; process
therefore, is no supply
the steady longer possible
of purine in
these obligate
nucleotides parasitesfor
is essential [47,48]. However,
survival. While the high expression
scavenging of nucleoside
these purines diphosphate
is the most kinase
energy efficient
required for the phosphorylation of adenosine and guanosine suggests that aspects
strategy, having an intact de novo purine synthesis pathway is highly advantageous to fungi that of purine
biosynthesis
inhabit still playwith
environments an important role in the metabolism
varying concentrations of purinesof[46].
these parasites [47].

Figure2.2.Structures
Figure Structuresofofthe
thekey
keyintermediates
intermediatesinvolved
involvedinindedenovo
novo and
and salvage
salvagepathways
pathwayscontaining
containingaa
purine ring (blue) and the resultant non-purine breakdown product
purine ring (blue) and the resultant non-purine breakdown product urea. urea.

6. Purine
Some Metabolism
species of fungi in Candida
have lostAlbicans
the ability to synthesize purines and rely solely on the salvage of
nucleotides from their environment. The Microsporidia have lost a number of enzymatic activities
While records detailing the symptoms of oral candidiasis date back to 400 B.C., for many
required for the de novo synthesis of purines, meaning the complete process is no longer possible in
centuries, these were thought to originate from the host rather than an infectious agent [49,50]. In
these obligate parasites [47,48]. However, the high expression of nucleoside diphosphate kinase
1771, Rosen von Rosenstein identified an invasive fungal pathogen as the causative agent of this
required for the phosphorylation of adenosine and guanosine suggests that aspects of purine
disease, and, in 1847, the French mycologist Charles Philippe Robin classified it as Oidium albicans
biosynthesis still play an important role in the metabolism of these parasites [47].
[51,52]. Almost a century later, Christine Marie Berkhout reclassified it under the current genus
6.Candida
Purine[52].
Metabolism in Candida Albicans
The commensal pathogen C. albicans is a frequent member of the gut microbiota; in healthy
While records
individuals, detailinginthe
it is observed symptoms of 40%
approximately oral candidiasis date back
of the population toFor
[25]. 400those
B.C., for
thatmany
do not centuries,
have an
these
intactwere thought
immune to originate
system, this from the host
pathogen rather
poses a than an infectious
major threat and agent
is [49,50].
the leadingIn 1771, Rosen
cause of
von Rosenstein identified an invasive fungal pathogen as the causative agent
hospital-acquired bloodstream infections, with those in intensive care units being most at risk [53]. of this disease, and, in
1847,
The the French
switch from mycologist
unicellularCharles Philippe
commensal Robin
yeast to classified
pleiomorphicit as Oidium
invasivealbicans
pathogen[51,52]. Almostby
is driven a
century
multiplelater, Christine Marie
environmental cues.Berkhout
In vitro,reclassified
this can be it under
induced theby current
changes genusin Candida [52].
pH, temperature, CO2
The commensal pathogen C. albicans
concentration, serum and many other factors [54–57]. is a frequent member of the gut microbiota; in healthy
individuals, it isform
The yeast observed in approximately
of C. albicans commonly40% of the
found population
in the [25]. Fortract
gastrointestinal thosehasthat do not access
plentiful have anto
intact immune system, this pathogen poses a major threat and is the leading cause
nutrients, including proteins, carbohydrates, fats and nucleotides such as purines. The concentration of hospital-acquired
bloodstream infections,
of available purines withdepending
varies those in intensive
on the dietcareofunits being(Table
the host most 1) at with
risk [53].
foodsThe switch
high from
in purines
unicellular commensal
such as seaweed yeast tomillimolar
containing pleiomorphic invasive pathogen
concentrations, and foods is driven
low byin multiple
purines suchenvironmental
as carrots
cues.
containing nanomolar concentrations [58]. Prior to purine absorption by the host in the many
In vitro, this can be induced by changes in pH, temperature, CO 2 concentration, serum and small
other factors
intestine – in[54–57].
particular, in the mucosa of the duodenum – these purines are available to be
The yeast
scavenged by form
the gut C. albicans commonly
of microbiota [59]. In its found in theform,
pathogenic gastrointestinal
C. albicans is tract has in
found plentiful access to
the bloodstream
nutrients, including proteins, carbohydrates, fats and nucleotides such as purines.
where it causes candidemia; the available purines in the blood are in the micromolar concentration The concentration
of available
range (Tablepurines
1) [60].varies depending on the diet of the host (Table 1) with foods high in purines such
as seaweed containing millimolar concentrations, and foods low in purines such as carrots containing
nanomolar concentrations [58]. Prior to purine absorption by the host in the small intestine—in
particular, in the mucosa of the duodenum—these purines are available to be scavenged by the
Microorganisms 2017, 5, 33 6 of 16

gut microbiota [59]. In its pathogenic form, C. albicans is found in the bloodstream where it causes
candidemia; the available purines in the blood are in the micromolar concentration range (Table 1) [60].

Table 1. Concentration of purines (µM unless indicated) from the habitats of C. albicans, A. fumigatus
and C. neoformans.

Source of purine Adenine Guanine Xanthine Hypoxanthine Inosine Reference

Average meal 1 (per gram) 0.9 1.0 1.8 0.02 ND [58]
Human Blood serum 0.4 97 20 172 168 [60]
Human Cerebral spinal fluid 0.2 0.5 2.4 3.9 0.6 [29,30]
Human Intracellular 1.5 97 ND 370 211 [60]
Plant matter average 2 0.4 µg/mL 1.3 µg/mL 0.8 µg/mL 1.0 µg/mL 1.2 µg/mL [61,62]
Soil average 3 19 M % 19 M % ND ND ND [63]
ND for no data. 1 Average meal defined as 100g rice, 75g carrot, 75g peas, 100g chicken [58]. 2 Average plant matter
concentration from of A. vaginalis, Z. jujuba, Z. jujuba var. spinosa and Z. mauritiana plants [61,62]. 3 An average soil
concentration was determined as proportion of purine relative to the g of air-dried soil from different locations [63].

Enzymes required for the degradation of purines into ammonia have not been well characterized
in C. albicans. BLASTp analyses using C. neoformans orthologs revealed that genes predicted to encode
the majority of purine degradation components are present in C. albicans. Six enzymes required for
the sequential breakdown of xanthine to ammonium were identified: xanthine oxidase (C2_00180C)
for the conversion of xanthine to uric acid, 5-hydroxyisourate (HIU) hydrolase (C2_08460C) for the
conversion HIU to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU), OHCU decarboxylase
(C3_01620W) for the conversion of OHCU to (S)-allantoin, allantoinase (C3_00180C) for the conversion
of allantoin to allantoate, and allantoicase (C2_00630C) converting allantoate to urea. For the final step
of hydrolyzing urea to ammonia, C. albicans encodes a urea amidolyase (C1_04660W) that carries out
two steps: carboxylation of urea to urea-1-carboxylate, followed by hydrolysis to two molecules of
ammonia. In addition, a zinc cluster transcription factor exclusively found in fungi has been identified
as playing a role in purine catabolism in C. albicans. Ppr1 regulates uracil degradation in S. cerevisiae but
plays a different role in C. albicans, where it is involved in the regulation of allantoin degradation [64].
Salvage of nitrogenous compounds by C. albicans from the environment is essential in the
production of virulence factors such as adherence to the host tissue, hyphal morphogenesis, and
release of ammonia to counteract acidification of the phagolysosome [65]. UV microscopy has shown
that, in vitro, C. albicans actively takes up the purines guanine and adenine within a few hours from the
growth medium. The concentrations of purines within the vacuole of the cell became supersaturated,
suggesting that when purines are available, the fungus scavenges all it can [66]. The consumption
of extracellular nucleotides in C. albicans occurs via their hydrolysis to nucleosides by ecto-enzymes
attached to the cell membrane that actively import purines into the cell [67].
While the enzymes of the purine salvage pathway have not been characterized in this species,
BLASTp analyses using C. neoformans orthologs revealed the presence of a gene predicted to encode
adenine phosphoribosyl transferase (C2_01430W), suggesting that C. albicans can covert adenine
to AMP. A likely member of the phosphoribosyl transferase family (C2_02740C) responsible for
the conversion of one or more of hypoxanthine, xanthine and/or guanine into their respective
phosphorylated nucleotides is also present. Other enzymes involved in the interconversion of purines
are anticipated in C. albicans; a BLASTp analysis using S. cerevisiae orthologs identified genes predicted
to encoded adenine deaminase (C2_03360W) and guanine deaminase (C7_00670W) for the conversion
of adenine to hypoxanthine and guanine to xanthine.
While only a few of the genes required for de novo purine biosynthesis have been characterized
in C. albicans, BLASTp analysis using C. neoformans orthologs reveals genes thought to encode
the ten enzymatic activities required for the conversion of phosphoribosyl pyrophosphate to IMP:
PRP transferase (C1_07710C), GAR synthetase/AIR synthetase (C1_07890C), GAR transformlyase
(C2_03090C), FGAM synthetase (CR_04740C), AIR carboxylase (C3_04520C), SAICAR synthetase
(CR_06150C), ADS lyase (CR_06150C) and AICAR transformylase/inosine cyclohydrolase (ATIC)
Microorganisms 2017, 5, 33 7 of 16

(CR_04090C). Additionally, the four enzymatic activities for synthesis of ATP from IMP (ADS
synthetase (C1_09640W), ADS lyase (CR_06150C), adenylate kinase (C6_01910W) and nucleotide
diphosphate kinase (C5_02890W)) and the four activities for GTP synthesis from IMP (IMP
dehydrogenase (C2_06390C), GMP synthase (C1_09490C), guanylate kinase (C503790W), and
nucleoside diphosphate kinase (C5_02890W)) are also predicted to be encoded by genes identified in
C. albicans.
Mutation and deletion studies of some of the genes encoding the enzymatic activities required
for de novo purine biosynthesis have been carried out in C. albicans. Mutants found to produce
red pigmentation were hypothesized to encode AIR carboxylase and SAICAR synthetase based on
S. cerevisiae studies identifying the pigmentation as a result of the accumulation of oxidized and
polymerized AIR in the vacuole [68]. Subsequent targeted gene disruption of the ade2 gene encoding
AIR carboxylase in C. albicans results in reduced virulence in a murine candidiasis model [69]. The
strain was unable to proliferate in human serum unless supplemented with exogenous adenine, and,
although not completely avirulent, exhibited a 100-fold attenuation of virulence [69].
The deletion of genes encoding two other enzymes of the de novo purine biosynthesis pathway
in C. albicans has also been performed. The genes ADE8 and GUA1 encoding the enzymes GAR
transformylase and GMP synthase have been deleted and in vivo growth assays for both strains
showed they were unable to grow on media without supplementation of exogenous purines; adenine
was required for the ade8 mutant and guanine was required for the gua1 mutant [70–72]. Both of
these deletion strains were hypersensitive to the purine biosynthesis inhibitors methotrexate and
6-azauracil [71]. In a candidiasis model of infection, the gua1 strain is avirulent [70].

7. Purine Metabolism in Aspergillus Fumigatus

Historically, the Aspergillus molds have been recognized as a genus since 1729 [2]. They have
been attributed to infection since the French revolution, and the species fumigatus known to frequently
cause aspergillosis since the early 1900s [73,74]. More recently A. fumigatus has become recognized
as the most prevalent airborne fungal pathogen, commonly causing severe or fatal infections in
immunocompromised individuals [75–77].
The asexual conidia of A. fumigatus are produced in abundance and inhaled by animals and
humans on a regular basis. In healthy individuals, these are cleared by the innate immune system;
however, in an immunodeficient individual, they pose a significant risk. Invasive aspergillosis is
frequently observed in cancer and transplant patients, accounting for 10–25% of life-threatening
opportunistic infections in leukemia treatment centers and 15–25% in transplant units [77–83].
Like many decomposers, A. fumigatus is commonly found in soil where organic matter provides
plentiful nutrients. Purine availability varies considerably in this niche depending on a number of
factors. Investigation of purine composition of soil has identified that humic acids (the principle
component of soil humus) are richer in the purines guanine and adenine than the pyrimidines cytosine,
thymine and uracil. The concentration of purines in dry soils have been shown to range from 21
to 138 µg per gram and the concentration distribution is consistent within soils: guanine is the
most abundant, followed by cytosine, adenine, thymine and the least abundant, uracil [63]. Purine
concentrations also vary greatly in plant life that will eventually become part of the diet of saprobes; the
legume Alysicarpus vaginalis and the Jujube fruiting tree species Ziziphus jujube and Ziziphus mauritiana
have been determined to have average purine concentrations of 0.005 to 2.6 µg/mL of guanine and
0.002 to 1.2 µg/mL of adenine (Table 1) [61,62].
Compared to this, the lung of an infected individual is a vastly different environment. The small
size of the conidia of A. fumigatus (2–3 µm) allows them to enter the respiratory tract, descend to
the aveoli and bind to surfactant proteins to be endocytosed by epithelial cells [78,84,85]. In the
lungs, host-produced extracellular ATP plays a role as an endogenous signaling molecule involved in
inflammation [86]. Once in the bloodstream, A. fumigatus encounters guanine at a concentration of
97 µM and adenine at 0.2 µM (Table 1).
Microorganisms 2017, 5, 33 8 of 16

While purine metabolism has not been well characterized in A. fumigatus, more extensive
characterization of the pathway in Aspergillus species has been carried out in Aspergillus nidulans,
with all enzyme-encoding genes believed to be associated with degradation, salvage, and de novo
biosynthesis of purines identified in this species [87–90]. BLASTp analysis using A. nidulans orthologs
revealed that the majority of purine degradation components are also likely present in A. fumigatus.
A. nidulans encodes a second enzyme, xanthine α ketoglutarate dependent dioxygenase, for the
conversion of xanthine to uric acid; this was not identified in A. fumigatus by BLASTp analysis but the
alternative enzyme xanthine oxidase (Afu2g10520) (which is also present in A. nidulans) was identified.
BLASTp analyses using A. nidulans orthologs revealed the genes predicted to encode enzymes
of the salvage pathway in A. fumigatus, including hypoxanthine xanthine guanine phosphoribosyl
transferase (Afu4g04550) and adenine phosphoribosyl transferase (Afu7g02310), suggesting that
A. fumigatus can convert hypoxanthine to IMP, xanthine to XMP, guanine to GMP, and adenine to AMP,
respectively. In addition to phosphoribosyl transferase enzymes, A. fumigatus is predicted to encode
adenine deaminase (Afu8g02860) and xanthine dehydrogenase (Afu4g11220) for the conversion of
adenine to hypoxanthine and hypoxanthine to xanthine. The adenine deaminase enzyme encoded by
the nadA gene is involved in the conversion of AMP to IMP and can be considered as a degradation
or a salvage enzyme. In A. nidulans, adenine deaminase is essential for the utilisation of adenine as a
sole nitrogen source, and, unlike other enzymes required for purine degradation, its expression is not
supressed by ammonium, perhaps reflecting an increase in purine interconversion when grown in
favourable conditions [91].
BLASTp analyses of the characterized genes of de novo purine biosynthesis from A. nidulans
predicted genes encoding the ten enzymatic activities required for the conversion of phosphoribosyl
pyrophosphate to IMP to be present in A. fumigatus, as are the four enzymatic activities for synthesis
of ATP from IMP and the four activities for synthesis of GTP from IMP. The deletion of the purine
biosynthesis GMP synthase-encoding guaA gene in A. fumigatus has shown that the strain is unable to
grow on media lacking exogenous guanine, and, in a murine model of infection, the guaA deletion
mutant was avirulent [70]. Computational modeling has supported the hypothesis that these purine
biosynthesis enzymes could serve as potential drug targets in A. fumigatus and the related species
Aspergillus niger [92].

8. Purine Metabolism in Cryptococcus Neoformans

The basidiomycete yeast Cryptococcus neoformans was first identified in 1894 by Sanfelice in peach
juice and associated with disease shortly after, identified from lesions from the tibia in a 31-year-old
patient [93,94]. The division of higher fungi or Basidiomycota is important for the effective breakdown
of organic compounds in the environment. Their coevolution with woody plants for over 350 million
years has given rise to many species possessing ligno-cellulytic enzymes that digest plant cell walls.
This digestive process is essential in the formation of soil humus [95–97]. The soil humus contains
varying levels of purines, highly dependent on the flora of the area (Table 1).
As well as soil, C. neoformans is commonly associated with bird guano. Unlike mammals, some
species require excess nitrogen to be converted to uric acid for its excretion rather than urea; the uric
acid cycle requires more energy but conserves water, which, for many organisms such as birds, is more
important [98,99]. Bird excreta, or guano, has long been valued for its fertilizing properties. Ancient
South American civilizations added this fertilizer to enrich soil and improve crops, risking their lives to
sail 21 km off the coast of Pisco to the guano-rich Chincha Islands [100]. This ancient tradition reached
Europe through the exploration of Alexander von Humboldt, leading to a time that became known as
the guano boom [101]. Chemists such as Gustav Magnus analyzed the nitrogen content to determine
the prices that guano should be sold. He reported an acid precipitation on the guano material and
found a novel compound now known as guanine to be in high concentrations [102]. Bird guano is
also high in uric acid, the ingredient responsible for the bird guano-associated damage of buildings,
Microorganisms 2017, 5, 33 9 of 16

particularly limestone. Since the identification of guanine from guano, no quantitative analysis has
been done to identify the specific concentration of purines in this substrate.
C. neoformans has been associated with bird guano since the 1960s and has since been identified
worldwide from bird droppings in a range of locations [21,22]. Pigeon guano medium supports the
growth of C. neoformans and the production of a key virulence factor, melanin. In addition, C. neoformans
is able to undergo its sexual cycle on pigeon guano, supporting the theory that pigeon guano is the
ecological niche of this fungal pathogen, as it can complete its life cycle solely in this environment [103].
Unlike Aspergillus species that can use a wide range of nitrogen sources, C. neoformans is more
limited and restricted to ammonium, amino acids and purines [37]. All genes encoding the predicted
enzymes of the purine degradation pathway in C. neoformans have been characterized. Six enzymatic
reactions are required for the breakdown of xanthine to ammonium and are as follows: urate oxidase for
oxidation of urate to HIU (CNAG_04307, URO1), HIU hydrolase (CNAG_06694, URO2) hydrolyzing
HIU to OHCU, OHCU decarboxylase (CNAG_00639, URO3) converting OHCU to (S) allantoin,
allantoinase (CNAG_00934, DAL1) hydrolyzing allantoin to allantoate, allantoicase (CNAG_01108,
DAL2) converting allantoate to urea, and finally urease (CNAG_05540, URE1) hydrolyzing urea to
ammonium [104]. Each of the genes identified to encode these enzymes has been sequentially deleted
and characterized [104]. None of these deleted genes affected production of the virulence factors
capsule or melanin, nor initiation of the C. neoformans sexual cycle [104]. In a murine inhalation model
of cryptococcosis, only urease, the final enzyme of the pathway for the hydrolysis of urea to ammonia,
is required for pathogenesis [105–107].
The salvage pathway in C. neoformans consists of a number of enzymes that can interconvert
purine intermediates. The nucleoside hydrolases are required for the hydrolysis of nucleotides
to nucleosides; hypoxanthine-xanthine-guanine phosphoribosyltransferase (CNAG_02546, HPT1)
(HXGPRT) and adenine phosphoribosyltransferase (CNAG_01390, APH1) phosphorylate the
nucleotides hypoxanthine, xanthine, guanine, and adenine to IMP, XMP, GMP and ATP,
respectively [108,109]. The phosphoribosyltransferases have been studied in this organism, and
the deletion of the genes encoding these enzymes did not result in any phenotypic differences from
the wild-type, nor affect virulence in a murine model of cryptococcosis suggesting purine salvage is
not important during the infection process [108,109].
Like C. albicans and A. fumigatus, C. neoformans encodes ten enzymatic activities for the conversion
of phosphoribosylpyrophosphate to IMP. Additionally, four enzymatic activities are required for IMP
to be converted to GMP and four enzymatic activities for its conversion to AMP. Deletion of the gene
encoding AIR carboxylase (CNAG_02294, ADE2) showed the identical phenotype to C. albicans and
S. cerevisiae of red pigmentation produced and similarly led to the pathway’s investigation as a potential
antifungal drug target. The ade2∆ mutant in a murine inhalation model and rabbit cryptococcal
meningitis model was avirulent [110,111]. Enzyme kinetic assays using recombinantly purified protein
revealed differences in activity between C. neoformans and Gallus gallus AIR carboxylase, suggesting
that this could be a novel target of inhibition against the fungal pathogen [112].
Analysis of the enzymes from the IMP branchpoint to either adenine or guanine
synthesis has been carried out for four enzymes in C. neoformans: adenylosuccinate synthetase
(ADSS) (CNAG_02858, ADE12), adenylosuccinate lyase (ADSL) (CNAG_03270, ADE13), inosine
monophosphate dehydrogenase (IMPDH) (CNAG_00441, IMD1) and guanine monophosphate
synthase (GMP synthase) (CNAG_01877, GUA1) [108,109,113,114]. Deletion of the genes encoding
these enzymes leads to strains that are purine auxotrophs, are attenuated for virulence factor
production and are avirulent in a murine inhalation model, contrasting starkly with the salvage mutants
and highlighting the importance of de novo purine biosynthesis during infection [108,109,113,114].
Biochemical and structural analyses have determined potential differences between these fungal
enzymes and their human counterparts that may be exploited in the development of fungal-specific
therapeutics [108,109,111–114].
Microorganisms 2017, 5, 33 10 of 16

9. Purine Biosynthesis as an Antifungal Drug Target

Since the 1940s, the synthesis of purines has been an important biochemical pathway in
the discovery of novel drugs [80,115,116]. The enzymatic activities of the purine biosynthesis
pathway have been particularly useful targets in the development or discovery of antibiotic,
anticancer and immunosuppressive agents, such as hadacidin, mercaptopurine, and mycophenolic
acid (MPA) [117–122].
MPA, an inhibitor of the rate-limiting enzyme IMPDH in de novo synthesis of
guanosine nucleotides, has been shown to have activity against C. albicans, A. fumigatus and
C. neoformans [70,108,123,124]. Mode of action studies have determined that MPA binds to the site of
the mobile flap of IMPDH and prevents formation of the closed enzyme conformation [125]. Studies
of C. neoformans IMPDH have shown that, while it is inhibited by MPA, unlike mammalian IMPDH,
this drug is able to bind to all conformations of the fungal IMPDH and not exclusively to the open
conformation [108]. Interestingly, only limited inhibition of A. fumigatus IMPDH by MPA occurs,
which is perhaps unsurprising given that MPA is produced by several Penicillium species commonly
found in the same environments, and A. fumigatus, therefore, may have developed some resistance to
the compound produced by competing species in its environmental niche [126]. Unfortunately, MPA
itself cannot be used as an antifungal agent in the clinic against opportunistic pathogens due to its
immunosuppressive activity; however, investigating this drug for activity against fungal enzymes
is proof of principle that the enzyme of these pathways can be targeted by inhibitors of purine
biosynthesis and may be a starting point for fungal specific inhibitor development.
The L-aspartate analog hadacidin has been identified as an antibiotic and anticancer agent
that targets ADS synthetase [117,127]. First isolated from Penicillium frequentans, this compound
exhibits 100% inhibition against Eschrichia coli as well as excellent clinical activity against human
adenocarcinoma [128]. However, in fungi, this compound does not show antifungal activity against
C. neoformans or A. fumigatus, and whilst there is some inhibition of C. albicans growth, this is
limited [109]. In enzyme kinetics assays, hadacidin cannot fully inhibit C. neoformans ADS synthetase,
but, again, this compound could serve as a basis for the development of antimycotics that act via
ADS synthetase [109]. While little, if anything, is known about the antifungal activity of other purine
biosynthesis inhibitors such as mercaptopurine (which closely resembles hypoxanthine and adenine
and targets the HGPRT enzyme), the known activities of MPA and hadacidin along with the available
crystal structures of their targets suggests that purine biosynthesis has the potential to be a valuable
target for future antimycotic development.

10. Conclusions
While the salvage of environmental purines, the synthesis of de novo purine nucleotides and the
breakdown of purines to their simplest form, ammonia, are common to the fungi that pose the most
consistent major threat to humans, these processes are not all essential during the infection process.
Scavenging purines from their environmental niche likely confers a selective advantage to A. fumigatus,
C. albicans and C. neoformans. However, during infection, the de novo biosynthesis pathway is essential,
likely due to pressures such as rapid proliferation, host immune defenses, and differences in purine
availability. Deletion or disruption of enzymes from the de novo purine pathway in all three species are
associated with either avirulence or reduced virulence of strains, making these an attractive antifungal
drug target.

Acknowledgments: Jessica L. Chitty is a recipient of a Queensland Medical Research Scholarship. James A. Fraser
received funding from the National Health and Medical Research Council, Grant APP1049716.
Author Contributions: Jessica L. Chitty and James A. Fraser wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Microorganisms 2017, 5, 33 11 of 16

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